The discovery that RNA molecules can function as enzymes revolutionized the understanding of chemistry in biological systems. It has now been demonstrated that RNA molecules can catalyze chemical transformations on themselves as well as on other RNA molecules [Castanotto, 1992]. The term ribozyme has been given to these RNA molecules. It has now become apparent that ribozymes play an important role in the biochemical function of many organisms.
Several types of ribozymes have been identified in living organisms. The first ribozyme to show catalytic turnover was RNA of ribonucleases P. Ribonucleases P (RNase P) cleaves precursor tRNAs (pre-tRNAs) at their 5' ends to give the mature 5'-termini of tRNAs . In Escherichia coli and Bacillus subtilis, the RNase P holoenzyme is composed of one basic protein subunit of approximate M.sub.r 14,000 (119 amino acids) and one single stranded RNA molecule of 377 and 401 nucleotides, respectively [Baer, 1990; Altman 1987; Waugh, 1989; Pace, 1990; Nichols, 1988]. The second ribozyme to show turnover was the L-19 intervening sequence (IVS) from tetrahymena. The 413 nucleotide intervening sequence (IVS) in the nuclear rRNA precursor from Tetrahymena thermophila can be excised and the two exons ligated in the complete absence of any protein [Kruger, 1982; Cech, 1981]. Unique to the Tetrahymena thermophila self-splicing reaction is the requirement of a guanosine or 5' guanosine nucleotide cofactor. The hammerhead self-cleavage reactions constitutes a third class of ribozymes. A number of plant pathogenic RNAs [Symons, 1989; Symons, 1990; Bruening, 1989; Bruening 1990], one animal viral RNA [Taylor, 1990] and a transcript from satellite II of DNA of the newt [Epstein, 1987; Epstein 1989] and from a Neurospora DNA plasmid [Saville, 1990] undergo a site specific self-cleavage reaction in vitro to produce cleavage fragments with a 2', 3'-cyclic phosphate and a 5'-hydroxyl group. This self-cleavage reaction is nonhydrolytic, unlike RNases P RNA cleavage of pre-tRNAs, where the internucleotide bond undergoes a phosphoryl transfer reaction in the presence of Mg.sup.++ or other divalent cations. Metal cations may be essential to RNA catalysis [Pyle, 1993]. Other reactions documented to date show that ribozymes can catalyze the cleavage of DNA [Robertson, 1990; Herschlag 1990], the replication of RNA strands [Green, 1992], the opening of 2'-3'-cyclic phosphate rings [Pan, 1992], as well as react with phosphate monoesters [Zaug, 1986] and carbon centers [Noller, 1992; Piccirilli, 1992]. Finally, ribozymes with new kinds of catalytic reactivity are being created through techniques of in vitro selection and evolution [Joyce, 1992].
The ability to specifically target and cleave a designated RNA sequence has led to much interest in the potential application of hammerhead ribozymes as gene therapy agents or drugs. One component to the success of treating a disease or targeting a specific RNA or DNA strand (substrate) to be cleaved is the optimization of the ribozyme/substrate complex. Optimization may be performed in vitro whereby a ribozyme is modified until it achieves maximum chemical activity on a target RNA or DNA molecule.
While much success has been achieved in vitro in targeting and cleaving a number of designated RNA sequences (Saxena and Ackerman, 1990; Lamb and Hayes, 1991; Evans, et al., 1992; Mazzolini, et al., 1992; Homann, et al., 1993), the translation of this success into ribozyme action in the whole cell has been limited. This is particularly the case for plant systems, with only one example of ribozyme induced cleavage presently reported (Steinecke, et al., 1992). Thus, it is not entirely clear how the success in vitro would correlate to the success in vivo.
Previous reports have demonstrated that high levels of ribozyme expression are required to achieve reduced accumulation of target sequence in vivo [Cameron and Jennings, 1989; Cotten and Birnsteil, 1989; Sioud and Drilca, 1991; LHuillier, et al., 1992; Perriman et al., 1993]. Additionally, a recent article suggests a necessity for the target and ribozyme to be sequestered in the same cellular compartment [Sullenger and Cech, 1993]. The results of several reports suggest that while the ribozyme molecule is clearly capable of inducing specific cleavage of a designated target RNA within a biological system, the rate limiting step, under in vivo conditions, is the formation of the active substrate/ribozyme hybrid. Such approaches towards optimizing the formation of the active substrate/ribozyme hybrid include attempts at "stabilizing" the ribozyme transcript by embedding the ribozyme within a stable RNA [Cameron and Jennings, 1989; Sioud, et al., 1992; Cotten and Birnsteil, 1989]. By providing the ribozyme transcript with a longer half-life, the chances of forming the desired hybrid with the target RNA are optimized.
In the approach of Cotten and Birnsteil (1989) a tRNA motif was used to deliver a hammerhead ribozyme to Xenopus oocytes and reduce the accumulation of a cytoplasmic target RNA. The embedded ribozyme was more effective than the analogous non-embedded ribozyme.